This invention relates to acridinium compounds and conjugates thereof. More particularly, the invention relates to acridinium compounds that are useful as chemiluminescent labelling agents because of their high emission efficiency and stability, as well as conjugates of such acridinium compounds and specific binding substances.
Acridinium esters have high emission efficiency and hence are useful as chemiluminescent labelling agents. In order for certain compounds to be practically useful as chemiluminescent labelling agents, it is generally essential that said compounds be highly stable in solution.
It is generally held that if the molecules of acridinium esters, taken as a whole, assume a planar structure on account of resonance, the strong electron withdrawing property of the acridinium ring causes electrons to be localized on the acridinium ring and, hence, the carbon atoms in the carbonyl group tend to become so much deficient of electrons that the esters are vulnerable to nucleophilic attack and, hence, labile. Under these circumstances, Unexamined Published Japanese Patent Application (kokai) Nos. Sho 63-101368 and Hei 3-501772 have disclosed acridinium esters that have an active group capable of binding with proteins and the like in the phenyl group or the 2-position of an acridinium group and which have a methyl group in the N-10 position, as well as a methyl group in the 2,6-position of the phenyl group.
Unexamined Published Japanese Patent Application (kokai) No. Hei 1-199949 teaches an acridinium ester having an active group capable of binding with proteins and the like in the N-10 position of the acridinium ring. The binding active group said acridinium ester has in the N-10 position is a bulky substituent such as a carboxymethyl group and, hence, steric hindrance is created between each of the hydrogen atoms in the 4,5-position of the acridinium ring and the substituents of nitrogen, rendering it difficult for the acridinium ring to assume a planar structure by itself; in this respect, the acridinium ester under consideration differs basically from the class of compounds that have a methyl group in the N-10 position and it would be advantageous from a stability viewpoint.
The acridinium esters which are taught in Unexamined Published Japanese Patent Application (kokai) Nos. Sho 63-101368 and Hei 3-501772 have another problem in that the functional group that has activity for binding with proteins and the like is bound to the benzene ring which is a leaving group, thereby putting considerable constraints when modifying the benzene ring. Further, the functional group having binding activity is highly reactive in itself and, hence, considerable difficulty is involved in the synthesis of these acridinium esters. In contrast, the acridinium ester which is taught in Unexamined Published Japanese Patent Application (kokai) No. Hei 1-199949 has activity for binding with proteins and the like in the N-10 position of the acridinium ring but it does not have any functional group for binding with proteins and the like in the leaving group. Therefore, it is fairly easy to modify the leaving group and, at the same time, the ester can be synthesized by a simple method since the only extra step that need be performed is to introduce the binding group at the final stage of synthesis. Because of these advantages, the acridinium ester under consideration has great utility in practical applications.
However, the acridinium ester compound that is specifically taught in Unexamined Published Japanese Patent Application (kokai) No. Hei 1-199949 is phenyl 10-carboxymethylacridinium-9-carboxylate bromide; since the benzene ring as a leaving group has no substituent, this compound has only low stability in solution and difficulty has been encountered with using it in practice as a labelling agents of specific binding substances.